Insulin is a polypeptide hormone secreted by the .beta.-cells of pancreas and takes part in regulating the blood sugar level. It consists of two peptide chains, i.e., A and B chains, which are linked by disulfide bridges at their cysteine residues and is produced by a proteolytic processing of proinsulin in pancreatic .beta.-cells(Insulin: Molecular Biology and Pathology, ed. Ashcroft, F. M. & Ashcroft, S. J. H., IRL Press, Oxford, 1992). Commercially, human insulin has been produced either by enzymatic process wherein an alanine residue at the 30th position of the B-chain of porcine insulin is replaced with a threonine residue through a transpeptidation reaction using trypsin(Markussen, J., Proceedings 1st International Symposium `Neue Insuline`, pp 38-44(1982), ed. Petersen, K. G., et al., Freiburger Greaphische Betriebe, Freiburg); or, by a process which uses genetically engineered E. coli(Chance, R. E., et al., Peptides: Synthesis-Structure-Function, pp 721-728(1981), in Proceedings of the Seventh American Peptide Symposium, ed. Rich, D. H. & Gross, E., Pierce Chemical Co., Rockford, Ill., U.S.A.; and Frank, B. H., et al., Peptides: Synthesis-Structure-Function, pp 729-738(1981), in Proceedings of the Seventh American Peptide Symposium, ed. Rich, D. H. & Gross, E., Pierce Chemical Co., Rockford, Ill., U.S.A.) or Saccharomyces cerevisiae(Thim, L., et al., Proc. Natl. Acad. Sci. U.S.A., 83, pp 6766-6770(1986); and Markussen, J., et al., Protein Engineering, 1, pp 205-213(1987)). As the enzymatic process for producing human insulin from porcine insulin is limited by its high cost, recent studies have been focused on processes for producing human insulin by genetic engineering techniques.
Chance et al. have reported a process for preparing insulin by: producing each of the A and B chains of insulin in the form of a fusion protein by culturing E. coli which carries a vector comprising a DNA encoding the fusion protein; cleaving the fusion protein with cyanogen bromide to obtain the A and B chains; sulfonating the A and B chains to obtain sulfonated chains; reacting the sulfonated B chain with an excess amount of the sulfonated A chain; and then, purifying the resultant to obtain insulin(Chance, R. E., et al., supra). However, this process has drawbacks in that it is cumbersome to operate two fermentation processes and the reaction step of the sulfonated A and B chains gives a low yield of insulin, making the process inherently impractical.
Frank et al. have reported a process for preparing insulin, which comprises: producing proinsulin in the form of a fusion protein by culturing E. coli which carries a vector comprising a DNA encoding the fusion protein; cutting the fusion protein with cyanogen bromide to obtain proinsulin; sulfonating proinsulin and separating the sulfonated proinsulin; refolding the sulfonated proinsulin to form correct disulfide bonds; treating the refolded proinsulin with trypsin and carboxypeptidase B; and, then, purifying the resultant to obtain insulin(Frank et al., supra). However, the yield of the refolded proinsulin having correct disulfide bonds sharply decreases as the concentration of proinsulin increases. This is due to the misfolding and some degree of polymerization involved and hence the process entails the inconvenience of using laborious purification steps during the recovery of proinsulin.
Thim et al. have reported a process for producing insulin in Saccharomyces cerevisiae, which comprises: producing a single chain insulin analogue having a certain amino acid sequence by culturing Saccharomyces cerevisiae cells; and isolating insulin therefrom via a series of steps, i.e., purification, enzyme reaction, acid hydrolysis and another purification(Thim, L., et al., supra). This process, although advantageous in that the purification procedures are relatively simple and no refolding procedure is necessary, still gives a low insulin yield, clue to the intrinsically low expression level of yeast system as compared to E. coli.
The role of the C-peptide in the folding of proinsulin is not precisely known. One of biochemistry textbooks describes that the C-peptide is necessary for the folding process to occur(Biochemistry, 3rd ed., p 41(1988), Freeman), but other studies have shown that about 30 to 50% of correctly folded insulin is obtainable by using the A and B chains alone in the absence of the C-peptide at very low concentration (Katsoyannis, P. G., et al., Biochemistry, 6, pp 2642-2655(1967); and Chance, R. E., et al., supra). It has also been shown that a high yield of correctly folded product, comparable to the yield obtainable by using proinsulin, can be obtained by using a peptide wherein the A and B chains are directly joined(Steiner, D. F. & Clark, J. L., Proc. Natl. Acad. Sci. U.S.A., 60, pp 622-629(1968); and Varandani, P. T. & Nafz, M. A., Arch. Biochem. Biophys., 141, pp 533-537(1970)). These results suggest that the role of the C-peptide is simply to bring the A and B chains closely together so as to facilitate the folding process.
According to the known three-dimensional structures of insulins in the Brookhaven Data Bank, the distance between C-terminal .alpha.-carbon of B chain(Thr-30) and N-terminal .alpha.-carbon of A chain(Gly-1) is roughly 5-11 .ANG. apart, which is a suitable distance for the insertion of a .beta.-turn structure. It has long been recognized that certain amino acid sequences have a high probability of being part of a turn conformation in proteins(Chou, P. Y. & Fasman, G. D., J. Mol. Biol., 115, 135-175(1977)), and this has more recently been shown to be true also for peptides in aqueous solution(Dyson, H. J., et al., J. Mol. Biol., 201, 161-200(1988); and Shin, H. C., et al., Biochemistry, 32, 6348-6355(1993)). Proline in the second position and glycine in the third position were found to give the highest .beta.-turn population, and an extensive study revealed that the nature of the amino acid at position 4 influences on the .beta.-turn stability in trans position, and there is a preference for a deprotonated Asp 4 side chain(Wright, P. E., et al., Biochemistry, 27, 7167-7175(1988); and Dyson, H. J., et al., supra).
.beta.-turns are likely sites for the initiation of protein folding, since they are determined by short-range interactions, they limit the conformational space available to the polypeptide chain, and by bringing more distance parts of the polypeptide chain together, they may be instrumental in directing subsequent folding events(Zimmerman, S. S. & Scheraga, H. A., Proc. Natl. Acad. Sci. U.S.A., 74, 4126-4129(1977); and Wright, P. E., et al., supra). These .beta.-turns are also known to play a valuable role in relation to an enzymatic cleavage.
Trypsin is a typical serine protease and hydrolyzes a protein or a peptide at the carboxyl terminal of an arginine or lysine residue(Enzymes, pp 261-262(1979), ed. Dixon, M. & Webb, E. C., Longman Group Ltd., London). In particular, facile hydrolysis occurs at a dibasic site where two successive arginine or lysine residues exist, and it is known that hydrolysis occurs most readily where the dibasic site is located in or next to a .beta.-turn structure(Rholam, M., et al., FEBS Lett., 207, 1-6(1986)).
As described above, there exists a need for a high-yield process for producing human insulin in a microorganism, and the present inventors have endeavored to develop an improved insulin production process and succeeded in establishing a new high-yield process by way of using a low molecular weight proinsulin derivative having an easily hydrolyzable .beta.-turn structure.